A set of TROSY-HNCO-based 3D experiments has been developed for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone 15N R1, R1rho, 15N-1H NOE, and 15N CSA/dipolar cross correlation has been demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsATM, is found to exhibit a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tc = 38 2.5 ns, at 50 C and a diffusion anisotropy, D// / D&#9524; = 0.79 0.05, corresponding to an aspect ratio a/b &#8805; 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S2 values in the 0.2-0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps-ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel.[unreadable] We also carried out an NMR study of the monomeric subunit of the channel (KcsAM), solubilized in SDS micelles. Chemical shift, solvent exchange, backbone 15N relaxation and residual dipolar coupling (RDC) data show the TM1 helix to remain intact, but the TM2 helix contains a distinct kink, which is subject to concentration-independent but pH-dependent conformational exchange on a microsecond time scale. The kink region, centered at G99, was previously implicated in gating of the tetrameric KcsA channel. An RDC-based model of KcsAM at acidic pH orients TM1 and the two helical segments of the kinked TM2 in a configuration reminiscent of the open conformation of the channel. Thus, the transition between states appears to be an inherent capability of the monomer, with the tetrameric assembly exerting a modulatory effect upon the transition which gives the channel its physiological gating profile.